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 Industrial Crops & Products 132 (2019) 413–429

Contents lists available at ScienceDirect

Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Antimicrobial activity of Eucalyptus camaldulensis Dehn. plant extracts and T

essential oils: A review
Verica Aleksic Sabo, Petar Knezevic
⁎

Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, Novi Sad, Vojvodina, Serbia

ARTICLE INFO ABSTRACT

Keywords: Eucalyptus has become one of the world’s most widely planted genera and E. camaldulensis (The River Red Gum)
Antimicrobial activity is a plantation species in many parts of the world. The plant traditional medical application indicates great
Plant extracts antimicrobial properties, so E. camaldulensis essential oils and plant extracts have been widely examined.
Essential oils Essential oil of E. camaldulensis is active against many Gram positive (0.07–1.1%) and Gram negative bacteria
Eucalyptus camaldulensis
(0.01–3.2%). The antibacterial effect is confirmed for bark and leaf extracts (conc. from 0.08 μg/mL to 200 mg/
mL), with significant variations depending on extraction procedure. Eucalyptus camaldulensis essential oil and
extracts are among the most active against bacteria when compared with those from other species of genus
Eucalyptus. The most fungal model organisms are sensitive to 0.125–1.0% of E. camaldulensis essential oil. The
extracts are active against C. albicans (0.2–200 mg/mL leaf extracts and 0.5 mg/mL bark extracts), and against
various dermatophytes. Of particular importance is considerable the extracts’ antiviral activity against animal
and human viruses (0.1–50 μg/mL). Although the antiprotozoal activity of E. camaldulensis essential oil and
extracts is in the order of magnitude of concentration several hundred mg/mL, it is considerable when taking
into account current therapy cost, toxicity, and protozoal growing resistance. Some studies show that essential
oils’ and extracts’ antimicrobial activity can be further potentiated in combinations with antibiotics (beta-lac-
tams, fluorochinolones, aminoglycosides, polymyxins), antivirals (acyclovir), and extracts of other plants (e.g.
Annona senegalensis; Psidium guajava). The present data confirm the river red gum considerable antimicrobial
properties, which should be further examined with particular attention to the mechanisms of antimicrobial
activity.

1. Introduction limited number of antiprotozoal and antiviral agents (El-Taweel, 2015;

The continuing increase in the degree of bacterial resistance to
conventional antibiotics is a problem of global significance. The crisis of
antimicrobial resistance has been ascribed to the misuse of these agents
and today resistant strains are common, such as methicillin-resistant
Staphylococcus aureus, vancomycin-resistant enterococci, drug resistant
Streptococcus pneumoniae and Mycobacterium tuberculosis, carbapenem-
resistant and extended-spectrum beta-lactamase producing en-
terobacteria, multidrug-resistant Pseudomonas aeruginosa and
Acientobacter baumannii etc. It was estimated that the medical cost per
patient with an antibiotic-resistant infection is up to $29,069 and in-
fections are usually life treating (Ventola, 2018; Aslam et al., 2018).
Similarly, fungi have become resistant to poliens, azoles and echino-
candins, and the emergence of drug-resistant strains has been reported
in all fungal species (Robbins et al., 2017). Beside the noticeable
emergence of resistant protozoa and viruses, there is a problem with
Irwin et al., 2016). This highlights the necessity for examination of new
antimicrobial agents and treatment strategies of infections caused by
the mentioned microorganisms.
The plant kingdom represents the source of various medicines.
Indeed, since ancient times medicinal plants play an important role of
health care population and could represent a significant source of new
antimicrobial drugs for combating pan- and multi-drug resistant mi-
croorganisms. These new antimicrobial agents could be hidden in
medicinal plant extracts and essential oils. One of the significant
medicinal plants is Eucalyptus camaldulensis. Thus, this review re-
presents the summary of previous researches data, regarding chemical
composition, antimicrobial activity, and other significant effects of
Eucalyptus camaldulensis.

⁎
Corresponding author.
E-mail address: petar.knezevic@dbe.uns.ac.rs (P. Knezevic).

https://doi.org/10.1016/j.indcrop.2019.02.051
Received 12 November 2018; Received in revised form 20 February 2019; Accepted 24 February 2019
Available online 05 March 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
V. Aleksic Sabo and P. Knezevic
1.1. Eucalyptus camaldulensis Dehn. (1832)
The River Red Gum (Eucalyptus camaldulensis Dehn.) is a tree be-
longing to the genus Eucalyptus from Myrtaceae family. This family
includes 140 genera and about 3800 species distributed in tropical and
subtropical regions of the world (Ali et al., 2011). The genus Eucalyptus
was described and named in 1788 by the French botanist l’Héritier. The
name is generic, from the Greek words ‘eu’ (well) and ‘kalyptos’ (cov-
ered), because the flowers of various Eucalyptus species are protected by
an operculum (Taoubi et al., 1997). The genus is indigenous to Aus-
tralia and Tasmania, consists of over 800 species, spreading worldwide
and successfully introducing due to its easy adaptability and fast growth
(Coppen, 2002). As a consequence, Eucalyptus has become one of the
world’s most widely planted genera (Akin et al., 2010) and a plantation
species in many parts of the world (Ames and Mathiews, 1968; Mubita
et al., 2008).
Eucalyptus camaldulensis (formerly Eucalyptus rostrata Schl.), also
known as long beak eucalyptus, murray red gum, red gum, river gum,
and red river gum, is one of the most widely distributed Eucalyptus
species. It is also considered one of the most widely planted trees in the
world (ca. 5 000 000 ha planted) (N.A.S., 1980; Boland et al., 1984).
Eucalyptus camaldulensis species is named for a private estate garden
near the Camaldoli monastery near Naples (L'Hortus Camaldulensis di
Napoli), from where the first specimen came to be described. Material
from this tree was used by Frederick Dehnhardt, Chief Gardener at the
Botanic Gardens in Naples, to describe this species in 1832 (Slee et al.,
2006).
The habitus of the plant is very specific. Life form of Eucalyptus
camaldulensis is a single-stemmed tree, with large trunk (Fig. 1). It is
medium-sized to tall tree, average height 30 m (Bren and Gibbs, 1986),
although some authors record trees to 45 m (Boland et al., 1984;
Brooker et al., 2002). Leaves are grey-blue, alternate, drooping,
8–22 cm long, 1–2 cm wide, often curved or sickle shaped, tapering, and
short pointed at base. Fruit is very small capsules at the end of thin
stalks, 5–8 mm, valves 4, and containing minute seeds. Eucalyptus ca-
maldulensis is an evergreen, perennial plant, and according to Jacobs
(1955) it could reach ages of 500–1000 years. Commonly grows on
riversides, whether of permanent or seasonal water (Brooker et al.,
2002), generally dominate in the community, forming pure open forests
or woodlands (Costermans, 1989).
The species Eucalyptus camaldulensis consists of two variations: E.
camaldulensis var. camaldulensis and E. camldulensis var. obtuse Blakely,
and one subspecies Eucalyptus camaldulensis subsp. simulata Brooker &
Kleinig (Table 1), found in North Queensland, which has been re-
cognised as a hybrid of E. camldulensis var. obtusa and Eucalyptus ter-
eticornis Smith (Brooker and Kleinig, 1994). According to the Centre for
Plant Biodiversity Research (EUCLID, 2006), the operculum shape in E.
Fig. 1. Eucalyptus camaldulensis (river red gum) on the Murchison River in Western Australia (by courtesy of Prof. Stephen D. Hopper).
414
Industrial Crops & Products 132 (2019) 413–429
camaldulensis is highly variable (Table 1). In the past, this character has
been used to break up the group into different varieties or subspecies.
The entire complex is currently under revision and new varieties or
subspecies may be described or extant ones rationalised. Until this work
is completed, EUCLID (2006) decided to adopt a conservative view of E.
camaldulensis.
1.2. Eucalyptus camaldulensis traditional and contemporary application
Used for centuries as a traditional Aboriginal herbal remedy, eu-
calyptus leaves and their essential oils have found various applications
in everyday life due to their antiseptic, anti-inflammatory and anti-
pyretic properties (Jeane et al., 2003; Kumar et al., 1988). Ancient
Aboriginal society in Australia used E. camaldulensis plant in medicines
to treat gastrointestinal symptoms (including colic, diarrhea, and dys-
entery), respiratory disease (colds, coughs, asthma, laryngalgia, lar-
yngitis, pharyngitis, sore throat, trachalgia), arrest bleeding, open
wounds, and cuts, as well as its decoctions for the relief of spasms,
aches, and pains in muscles, but also pains in joints and even tooth
(Duke and Wain, 1981).
As previously stated, E. camaldulensis plant is also known as red gum
eucalyptus, murray red gum, and river red gum, because it produces red
kino i.e. red gum, in significant amount. Thus besides different plant
parts Aborigines used its secondary products as folk remedies. They
made incisions in the tree trunks for obtaining the red kino and applied
it directly to abrasions and cuts. Except fresh kino, the dried, dehy-
drated kino was prepared and used in the same way as fresh, but after
softening in water (Williams, 2011; Clarke, 2014). Another significant
folk remedy is young leaves which were used for smoke bath, where
burning leaves smoke surrounds patient. The smoking medicine was
used for fevers, colds, flu and general sickness (Williams, 2011;
Pennacchio et al., 2010; Duke and Wain, 1981). Being useful for
treating various health conditions, E. camaldulensis and its folk remedies
then were transferred and introduced to other parts of the world, such
as Africa. In Sudan the red kino was used for sore throat and diarrhea,
while the smoke of burnt leaves was inhaled in case of respiratory
problems. In Senegal for stomach-ache decoctions from leaves were
prepared with sugar, while in Zimbabwe for cough, flu, and fever a
decoction of E. camaldulensis leaves were combined with Citrus limon
(L.) Burm. f. fruits and Psidium guajava L. leaves (Doran and Wongkaev,
2008; Maroyi, 2013). Further more, to prevent tooth decay and peri-
odontitis in Nigeria teeth cleaning sticks were made from tree (Bukar
et al., 2004), and in traditional medicine for healing wound infections,
poultice of leaves containing eucalyptus oil have been used (Adeniyi
et al., 2006).
Nowdays, E. camaldulensis has been the subject of numerous studies,
to confirm plant’s usefulness in traditional medicine for the treatment
V. Aleksic Sabo and P. Knezevic Industrial Crops & Products 132 (2019) 413–429

Table 1
The main characters that distinguish Eucalyptus camaldulensis taxa.
Taxon Bark Leaves Operculum

E. camaldulensis var. grey-white, rough at non-glaucous, green, narrowly strongly beaked 0.3-0.7 cm long
camaldulensis base bark lanceolate juvenile leaves

E. camaldulensis var. essentially white,
obtusa smooth and seasonally
powdery bark
E. camadulensis subsp. grey-white, smooth to
simulata base bark
dull to slightly glossy green adult
leaves with densely to very
denselyreticulate venation
ovate or lanceolate juvenile leaves,
adult leaves with dense reticulation
rounded or obtusely conical
operculum 0.4-0.7 cm long at
maturity and glaucous juvenile
growth
long horn-shaped operculum
0.9–1.6 cm long

of various ailments (Coelho-de-Souza et al., 2005; Ghani, 2003; Ito
et al., 2000). Its essential oils are reported to be anesthetic, antiseptic
and astringent (Jeane et al., 2003; Kumar et al., 1988). In addition, a
decoction of the leaves is reported to be a remedy for sore throat and
other bacterial infections of the respiratory and urinary tracts
(Bruneton, 1999). Due to numerous contemporary data regarding the
antimicrobial activity of E. camaldulensis, this subject will be discussed
in special section/chapter.
Except antimicrobial effects, E. camaldulensis plant extracts (PEx)
and essential oils (EOs) and its constituents possess numerous other
beneficial effects. One such effect is certainly gastrointestinal effect. In
animal models, extracts of the leaves of E. camaldulensis and E. torelliana
R. Muell are reported to decrease gastric acid production and thus ap-
pear useful for the treatment of gastric ulcers (Adeniyi et al., 2006). It
has been proven that E. camaldulensis leaves methanol extracts pos-
sessed ulcer-healing promoting effect when investigated in acetic acid
induced-ulcer in rat (Rattus norvegicus domesticus) (Lawal et al., 2014)
(Table 2). Similarly, the poultice of the leaves is applied over wounds
and ulcers (Gill, 1992).
The antioxidant effect represents one more useful E. camaldulensis
characteristic. The free radical scavenging activities of the essential oils
is assessed by measuring their scavenging abilities for 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radicals. The scavenging activity for the
Eucalyptus camaldulensis was characterized as high (81.9%) (Ghaffar
et al., 2015). The results of E. camaldulensis EO antioxidant effect eva-
luation indicated that the EO had high potent ferrous ions chelating and
total antioxidant activities comparing to ascorbic acid and BHT (El-Baz
et al., 2015). Also, the E. camaldulensis var. brevirostris leaves ethanol
extract possessed antioxidant activity, where the prevailing anti-
oxidants in the extract were gallic and ellagic acid (El-Ghorab et al.,
2003). There are several more studies dealing with antioxidant activity
of E. camaldulensis (Barra et al., 2010; Salem et al., 2015; Siramon and
Ohtani, 2007; Olawore and Ololade, 2017), but it is interesting to
mention here that E. camaldulensis flower EO inhibited melanogenesis
through its antioxidant properties and by down-regulating both mi-
togen-activated protein kinases (MAPK) and protein kinase A (PKA)
signaling pathways. This study indicated that the essential oil has the
potential to be developed into a skin care product (Huang et al., 2015).
Thus, except the medical application, E. camaldulensis extracts are also
currently used in cosmetic formulations, and leaf extracts have been
approved as food additives (Takahashi et al., 2004). Also, essential oils
and their constituents have been used as flavoring agents in the for-
mulation of different pharmaceutical products, cosmetics, and food
industry (Cowan, 1999). Except this, E. camaldulensis PEx and EOs have
the potential to be used as antibacterial and antifungal agents in cos-
metic and pharmaceutical products. It is interesting to mention here the
activity of E. camaldulensis EOs (2–8 mg/mL) on dental biofilm forma-
tion in vivo where detected inhibition was 14.5–39.2% after four weeks,
comparing to chlorhexidine (2 mg/mL), which inhibited maximum
13.9% of dental biofilm formation (Rasooli et al., 2009). Extracts and
essential oils of this aromatic plant can be used as food preservatives in
order to reduce the dependency on synthetic chemicals in food pre-
servation. In Australia, it is also used as sources of wild honey, pro-
viding bees with good quality pollens and heavy yields of nectar
(Boland et al., 1984). Moreover, in industry, the wood of E. camaldu-
lensis has been used for heavy construction, railway sleepers, flooring,
framing, fencing, plywood, and veneer manufacture, wood turning,
firewood, and charcoal production (Boland et al., 1984). Eucalyptus
camaldulensis wood burns well and make a good fuel, also its dense
wood and coppicing ability make it an excellent species for fuelwood
production used in several countries such as Brazil (Jacobs, 1981;
Eldridge et al., 1993). Furthermore, Eucalyptus biomass residues from
agro-forest and pulping industries represent a valuable source of high-
value compounds such as triterpenic compounds (Domingues et al.,
2011; Ferreira et al., 2018).
Due to the diversity of E. camaldulensis beneficial effects, all effects
reported in the literature are summarized in Table 2. Beside the bene-
ficial effects, essential oils and plant extracts can exert potentially un-
favorable effects as complex mixtures of different compounds. A risk
assessment of their hazard is always necessary before commercializa-
tion, so the estimation of EOs toxicity has been already conducted re-
sulting in human oral and dermal dose limit recommendations. For
most EOs the recommended dose is in range 1–4%, but for cineole-rich
Eucalyptus sp. EOs, including E. camaldulensis, the limit dose is 10%,
indicating a generally low application risk (Lis-Balchin, 2006). The safe
daily oral dose in human adults is 300–600 mg, while semisolid pre-
parations for topical use may contain 5–20% of Eucalyptus oil
(Blumenthal and Busse, 1998; Tisserand and Young, 2014). Similarly, E.
camaldulensis bark methanolic extract LD50 value for Swiss albino mice
(Mus musculus) is very high – 1120 mg/kg, indicating its low host toxic
effects (Islam et al., 2014). The low toxicity and natural origin of E.
camaldulensis essential oil and extracts, in contrast to synthetic anti-
microbials, favor their application as antimicrobial agents.
2. Chemical composition
2.1. Eucalyptus camaldulensis plant chemical composition
Eucalyptus camaldulensis leaves contain 0.1–0.4% essential oil, of
which 77% is 1,8-cineole. There is considerable amount of cuminal,
phellandrene, aromadendren (or aromadendral), valerylaldehyde, ger-
aniol, cymene, and phellandral (Council of Scientific and Industrial
Research, 1948–1976Council of Scientific and Industrial Research
(S.I.R, 1948Council of Scientific and Industrial Research, 1948–1976;
Slee et al., 2006). Leaves contain 5–11% tannin. The kino (a class of
wood exudates), contains 45% kinotannic acid as well as kino red, a

415

Table 2
Different significant effects of Eucalyptus camaldulensis.
Different E. camaldulensis effects
Gastrointestinal effect
Anti-inflammatory and analgesic
effect
Anti-nociceptive activities
Cytotoxic effect
416
Anti-parasitic, insecticidal and
repellent effects
Anti-diabetic effect
Model organism and/or cell line
Acetic acid induced-ulcer in rat
Four ruminal fistulated buffaloes (Syncerus
caffer), 4 years old with initial body weight
321 ± 20 kg
Healthy albino rats (200 ± 30 g)
Healthy albino rats (200 ± 30 g)
Two human breast cancer cell lines (MCF 7 and
MDA-MB-231)
L20B (a genetically engineered mouse cell line)
and human rhabdomyo sarcoma cells
Ehrlich's ascites carcinoma (EAC) in Swiss
albino mice
Trypanosoma brucei infected mice
Promastigotes of Leishmania major in vitro
Larvicidal activity against Anopheles stephenssi
Mosquito larvicidal against two mosquito
species, Aedes aegypti and Aedes albopictus
Aphis gossypii Glover (Hem: Aphididae)
Repellency against the adult females of Culex
pipiens
Albino rats
Alloxan-induced diabetic rats
Plant part extract, oil or compound
Leaves methanol extracts
Leaves meal
Seed essential oils of E. camaldulensis
var. nancy and E. camaldulensis var.
petford
Seed essential oils of E. camaldulensis
var. nancy and E. camaldulensis var.
petford
Leaves methanol, ethyl acetate, n-
buthanol, and water extracts
Crude methanol extracts
Stem bark methanol extract
Leaves, stem and root barks extracts
Methanol and aqueous extracts
Leaves extract and volatile oil
Leaves essential oils and their 12
constituents
Essential oil
Dried fruits essential oil
Leaves ethanol extract
Leaves ethanol extract
V. Aleksic Sabo and P. Knezevic
Effect of dosage and/or application mode Reference
Reduction the size from day 5 in animals treated with 500 mg/kg body weight of Lawal et al. (2014)
reconstituted extracts at 24 h interval
Dietary treatments - different levels of leaf meal supplementation at 0, 40, 80, and Thao et al. (2015)
120 g/hd/d for 21 days.
Protozoa count and proteolytic bacteria population were reduced (p < 0.05).
Fungal spores, amylolytic, cellulolytic and total viable bacteria were unchanged.
Carrageenan induced paw oedema test model in rats, which received 1000 μg kg−1 Olawore and Ololade
body weight; 43.75-87.5% of anti-inflammatory activity (2017)
Rats treated with 1000 μg kg−1 body weight, measured neurogenic and Olawore and Ololade
inflammatory pain responses, 41.03-99.09% of inhibition (2017)
Significant cytotoxic potential with IC50 values ranging from 3 to 250 μg/mL after Hrubik et al. (2012)
72h
Moderate cytotoxicity Adeniyi et al. (2015)
25, 50 and 100 mg/kg/day for 5 days; High LD50 value (1120 mg/kg) Islam et al. (2014)
200-600 mg/kg body weight/day of the hexane, ethyl acetate, methanol and water Kabiru et al. (2013)
extracts for 21consecutive days
IC50 values were 586.2 ± 47.6 and 1108.6 ± 51.9 μg/mL Nosratabadi et al.
(2015)
LC50 values of 89.85 and 397.75 ppm, respectively. Clear dose-response Medhi et al. (2010)
relationships, the highest dose of 320 ppm essential oil extract resulted almost in
100% mortality in the population after 24 h of exposure
400, 200, 100, 50, and 25 μg/mL of essential oil were tested and each compound Cheng et al. (2009)
was tested at 50, 25, 12.5, and 6.25 μg/mL; Mortality was recorded after 24 h;
LC50 values 31.0-55.3 lg/mL, LC90 values 71.8-192.4 lg/mL.
LC50values 2.28 μl L−1air Ebrahimi et al. (2013)
Two different treatment levels (5 and 10 μl) in six exposure times (15, 75, 135, Erler et al. (2006)
195, 255 and 315 s)
Oral 500 mg/kg of body weight Dawoud (2015)
500 mg/kg of body weight in distilled water orally, E. camaldulensis leaves Dawoud and Shayoub
supplement incorporated into the diet 5 g/kg/day (2017)
Industrial Crops & Products 132 (2019) 413–429
V. Aleksic Sabo and P. Knezevic Industrial Crops & Products 132 (2019) 413–429

glucoside, catechol, and pyrocatechol. Leaves and fruits test positive for
flavonoids and sterols. The bark contains 2.5–16% tannin, the wood
2–14%, and the kino 46.2–76.7% (Watt and Breyer-Brandwijk, 1962).
Some of the reported phytoconstituents of the tree included essential
oils, sterols, alkaloids, glycosides, flavonoids, tannins, and phenols.
2.2. Eucalyptus camaldulensis essential oil chemical composition
A considerable variation in the yield of leaf essential oil from E. ca-
maldulensis has been reported (Boland et al., 1984; Shieh, 1996;
Moudachirou et al., 1999; Farah et al., 2002), depending on multiple
biotope factors, and also genetic and/or epigenetic characteristics of the
plant. The yields of E. camaldulensis leaves EO (0.90–0.98%) originating
from Pakistan and Morocco were similar (Ashraf et al., 2010; Farah et al.,
2002), while Moudachirou et al. (1999) reported a variable oil content of
0.6–1.4% from different locations of Benin. The oil yield of E. camaldu-
lensis from Jerusalem was 0.5% (Chalchat et al., 2000) and significantly
higher oil yield was reported for E. camaldulensis from Taiwan: 2.3–3.0%
with respect to different seasons (Shieh, 1996). Similar EOs yield
(0.77–2.53%) has been reported for E. globulus, as one of the econom-
ically important plants for essential oil production (Joshi, 2012;
Selvakumar et al., 2012; Harkat-Madouri et al., 2015). The reported es-
sential oil yield for other Eucalyptus species is slightly higher, ranging
from 1.2% to 3% (w/w): the highest yield was obtained from E. cinerea F.
Muell. ex Benth and E. sideroxylon A. Cunn. ex Woolls (3.0%), followed
by E. lehmannii (Schauer) Benth. (2.8%), E. bicostata Maiden, Blakely &
J.H.Simmonds (2.0%), E. leucoxylon F. Muell (1.6%), E. maidenii F.Muell.
(1.5%), and E. astringens Maiden (1.2%) (Sebei et al., 2015).
All the E. camaldulensis essential oil single compounds belong to
chemical class of hydrocarbons terpenes, further devided according to
the number of isoprene units (C5H8) to monoterepenes (C10H16), ses-
quterepenes (C15H24), and longer chains of isoprene units. Oxygenated
terpenes (oxygenated monoterepenes and oxygenated sesquterepenes)
are called trepenoids. According to the literature, in E. camaldulensis
EOs dominates 1,8-cineole (eucalyptol), trans-pinocarveol and terpinen-
4-ol from chemical class of oxygenated monoterepenes. The oils also
contain considerable amount of monoterpene hydrocarbons (β-pinene,
α-thujene, γ-terpinene, p-cymene), while sesquterepene hydrocarbons
and oxygenated sesquterepenes are detected in significantly lower
amounts (Table 3).
When the composition of five Eucalyptus essential oils (Eucalyptus
camaldulensis, Eucalyptus astringens Maiden, Eucalyptus leucoxylon,
Eucalyptus lehmannii and Eucalyptus rudis Endl) are compared, a high
percentage of monoterpenes, mainly oxygenated compounds, with
lower quantities of sesquiterpenes were recorded throughout the four
seasons (Ben Jemaa et al., 2012). In E. camaldulensis oil, monoterpenes
were prevalent (34.6–56.3% for all seasons) while sesquiterpene hy-
drocarbons (6.6–16.5%) and oxygenated sesquiterpenes (2.1–11.1%)
were present in less extent. Eucalyptus astringens oil had resembling
chemical characteristic to E. camaldulensis oil, the abundant quantity of
monoterpenes, which represented more than 50% of the total oil
amounts for the all seasons (53.4–63.6 %). Similarlly, E. leucoxylon
essential oil, contained mainly oxygenated monoterpenes with a pre-
valence of 1,8 cineole (13.1–17.6%), such as in E. camaldulensis oil
(15.5–20.6%). The essential oil of E. lehmannii also was made up largely
of monoterpenes (hydrocarbons 27.6–44.9% and oxygenated
30.9–62.8%), with smaller amounts of sesquiterpene hydrocarbons and
absence of oxygenated sesquiterpenes, unlike the oils of other Eu-
calyptus species (Ben Jemaa et al., 2012).
The difference in the chemical composition of the E. camaldulensis
EOs may be due to many reasons, which can generally be classified in
five main groups: (1) the change in plant genes through generations and
hybridizations (naturally and induced) may result in production a
variety of volatile oils compared with those of different habitat; (2)
nutrients of different soils and their accumulation in the leaves may
result in different plant metabolism and consequently production of
different bio-products and also EOs made of diverse compounds in
variable amounts; (3) acclimation of species to the Australian en-
vironment in which it is growing in the past, compared with the in-
troduced and/or worldwide planted trees on plantations; (4) different
ecotypes of the E. camaldulensis and (5) differences may be due to plant
part used for essential oils extraction and its stage of development
(maturity). Knowing that these factors express significant effect on the
percent compositions of some EO components for this species, E. ca-
maldulensis can be grown in the corresponding areas and in specific
conditions to enhance EO production.
The variations in the chemical composition of eucalyptus EOs with
respect to seasons have also been reported (Tsiri et al., 2003), but the
most commonly detected as major components in the eucalyptus es-
sential oil are 1,8-cineole, β-pinene, γ-terpinene, and p-cymene. It is

Table 3
Classes of major Euclyptus camaldulensis essential oils compounds and their content in different plant parts.
Chemical classes Chemical subclasses Sublaclasses content in leaf and fruit (%)a Major essential oils compounds Major compounds content in oila,b

L Fr Fl

Terpenes Monoterpene hydrocarbons 5.7-52.2 L α-Pinene 1.7-28.3 1.12-3 3.51

18.7 Fr β-Pinene 0.3-18.6 8.8 7.7-27.09
18.5 Fl α-Thujene 1.0-3.4 0.3 0.6-0.77
β-Phellandrene 0.5-7.5 0.3 2.1-2.2
p-Cymene tr. -6.5 4.8 9.32
γ-Terpinene 0.19-7.6 0.2 0.4-0.5
Sesquiterpene hydrocarbons 1.8-3.6 L Aromadendrene 0.1-11 0.2 0.21-0.24
6.1 Fr allo-Aromadendrene 0.2-1 3 0.2
1.2 F%l Bicyclogermacrene 0.3-1.8 2 0.09-0.4
α-Copaene 0.9 tr. –

Terpenoids Oxygenated monoterpenes 40.8-87.42 L 1,8-Cineole 13.73-84.9 3.8 34.7-69.26

14.1 Fr trans-Pinocarveol 0.06-8.5 0.2 0.11-1.9
50 Fl Terpinen-4-ol 0.27-5.2 1.9 3.29-3.6
Myrtenol 1.4-9.75 2.1 0.12
Cuminal 0.09-3.2 1.3 0.94-1.01
Oxygenated sesquiterpenes 4.9-39.6 L Spathulenol 0.16-19.2 19 0.12-10.18
23.2 Fr Elemol 0.6-3 – –
12.7 Fl β-Eudesmol 0.13-4.4 – –
cis-Farnesol 0.9-5 – 0.1

a
L- leaf, Fr-fruit, Fl-flower.
b
tr., traces (< 0.05%).

417
V. Aleksic Sabo and P. Knezevic
important to emphasize here that according to chemical composition
the Eucalyptus camaldulensis EO generally can be divided in two dif-
ferent types. Type I is a cineole-rich essential oil containing 80–90%
1,8-cineole plus pinene, and Type II is a cineole-poor essential oil,
containing significantly less cineol (Williams, 2011). Plant genotype is
important factor influencing the final chemical composition of EO
(Djilani and Dicko, 2012). Due to genetic and epigenetic factors same
plant species can produce a similar EO, but with different chemical
composition and therapeutic activities. Brophy and Southwell (2002)
examined essential oils of two variations, Eucalyptus camaledulensis var.
camaldulensis and Eucalyptus camaledulensis var. obtuse, and the main
compounds of Eucalyptus camaledulensis var. camaldulensis were p-
cymene (22%), cryptone (14%), and spathulenol (17%), while Eu-
calyptus camaledulensis var. obtusa had different main compounds: 1,8-
cinole (52%), α-pinene (15%), and aromadendrene (3%), suggesting
that these subspecies belong to different types of eucalyptus essential
oils. The content of 1,8-cineole was also variable but in the same range
for essential oils reported from Greece (25.3–44.2%) (Tsiri et al., 2003),
Pakistan (34.4–40.0%) (Ashraf et al., 2010), Mozambique
(37.1–40.0%) (Pegula et al., 2000), Nigeria (32.8–70.4%) (Oyedeji
et al., 1999), and Taiwan (34.0–68.2%) (Shieh, 1996). Similarly, the
major component of Eucalyptus camaldulensis oils originating form
Burundi, Morocco, and Benin was 1,8-cineole ranging from 31.0 to
72.5% with no presence of cryptone (Dethier et al., 1994; Zrira and
Benjilali, 1991; Zrira et al., 1992; Moudachirou et al., 1999). Cryptone
has been shown to be present in low-cineole varieties of EOs from
Australia (Bignell et al., 1996), Uruguay (Dellacassa et al., 1990) and
South Florida (Pappas and Sheppard-Hanger, 2000). For example, the
major constituents identified in the essential oil from South Florida
included p-cymene (35.0%), cryptone (13.7%), terpinen-4-ol (5.7%),
spathulenol (4.3%), and cuminaldehyde (3.7%), with a very low
amount of 1,8-cineole (2.7%) (Pappas and Sheppard-Hanger, 2000).
It was proven that essential oils of different plant parts have dif-
ferent chemical composition (Table 3). Previous studies on the essential
oil of E. camaldulensis flowers revealed the presence of 1,8-cineole, β-
pinene, and spathulenol as the most abundant constituents (Giamakis
et al., 2001). The essential oil of the leaves was found to contain p-
cymene, γ-terpinene, α-pinene, 1,8-cineole, terpinen-4-ol, α-terpineol,
carvacrol, and thymol as the major components (Siramon and Ohtani,
2007). The major components of the fruits essential oil were ar-
omadendrene, α-pinene, drimenol, and cubenol (El-Ghorab et al.,
2002).
Plant developmental stage and maturity of plant parts used for es-
sential oil extraction also affect essential oil chemical composition.
Giamakis et al. (2001) analyzed the immature flowers and calli grown
in Athens (Greece), and they found that the main monoterpenes pro-
duced in E. camaldulensis calli, cultured in darkness and under light
conditions, were 1,8-cineole, 62.70 and 69.26% as well as β-pinene,
27.09 and 25.31%, respectively. In lower amounts α-pinene (1.2 and
1.1%, respectively) and the terpenoids, camphene, myrcene, isocineole,
myrtenol, bicyclogermacrene, spathulenol, and trans-pinocarveol were
present (less than 1%). Apart from isocineole, these constituents were
also determined in the essential oil from immature flowers. This is re-
markable because Giamakis et al. (2001) showed that undifferentiated
calli are capable of producing high amounts of monoterpenoid com-
pounds (approx. one third of that produced by the explant). This makes
immature flowers from E. camaldulensis an interesting candidate for the
development of calli able to produce high percentages of these two
important monoterpenes, 1,8-cineole and β-pinene (Giamakis et al.,
2001). Also, the influence of light conditions on essential oil chemical
composition showed that light conditions did not considerably affect
the production of monoterpenes and sesquiterpenes compounds by calli
developed from immature flowers (Giamakis et al., 2001).
Furthermore, plant culture conditions can influence the essential oil
chemical composition. Soil salinity is a key ecological stress that se-
verely influences plant productivity (Williams, 2011). Ashraf et al.
418
Industrial Crops & Products 132 (2019) 413–429
(2010) showed that salinity had a considerable effect on the percent
compositions of some components of E. camaldulensis leaves essential
oil. The mean values of 1,8-cineole content of EO from saline and non-
saline provenances of Pakistan were 34.42 and 40.05%, respectively
(Ashraf et al., 2010). Therefore, they recommend stressing of this spe-
cies by growing in the saline areas to enhance essential oil production
for its various medicinal and pharmacological uses.
2.3. Eucalyptus camaldulensis extracts chemical composition
Extraction represents the primary step in obtaining the crude mix-
ture of compounds from plants. Quality and quantity of the extracts
dependent of the target compound structures, natural sources, and type
of processes (Karacabey et al., 2013), explaining the different phenolic
composition in the extracts obtained with different procedures. The
most commonly plant extract have been obtained by conventional
solvent extraction methods (infusion, decoction, digestion, maceration,
and percolation) (Azwanida, 2015) using solvents such as water,
ethanol, methanol, chloroform, dimethyl-sulfoxide etc. However, these
techniques are demanding regarding the extraction process duration,
organic solvent consumption, and lack of extraction automation. The
potential and powerful alternative to conventional liquid solvent ex-
traction methods are Ultrasonic-Assisted Extraction (UAE) and Micro-
wave-Assisted Extraction (MAE), especially in the case of plant material
(Hao et al., 2002; Eskilsson and Bjrklund, 2000). Interest in MAE has
increased significantly over the past 5–10 years in particular medicinal
plant research, as a result of its inherent advantages as special heating
mechanism, moderate capital cost, and its good performance under
atmospheric conditions (Ballard et al., 2010; Chan et al., 2011). In
addition, MAE technique possesses many advantages compared with
other methods for the extraction of compounds such as bioactive
compounds (Sanchez-Aldana et al., 2013) saving in processing time and
solvent, higher extraction rate, better products with lower cost, reduced
energy consumption (up to 85-fold savings), and waste generation (Yan
et al., 2010; Yemis and Mazza, 2012). This is confirmed for E. ca-
maldulensis extraction of phenolic and flavonoid compounds, where the
compounds were extracted with MAE for 5 min which was equivalent
with UAE (60 min) and traditional extraction (24 h) methods
(Gharekhani et al., 2012).
Many authors reported the chemical composition of E. camaldulensis
extracts. The leaves of E. camaldulensis from the zoo-botanical garden in
Giza (Egypt) yielded 4 major fractions (Singab et al., 2011). The major
components of the first fraction (eluted with water) were identified as
HHDP-glucopyranose, chlorogenic acid, and phloroglucinol derivatives.
The second 30% methanol fraction was found to contain different
galloyl-HHDP-glucopyranose positional isomers and pedunculagin as
major components. The third 60% methanol fraction was pre-
dominantly composed of digalloyl-HHDP-glucopyranose (tell-
imagrandin I) α and β anomers, while the last 100% methanol fraction
was composed of a mixture of ellagitannin dimers. The profiling of the
obtained fractions by HPLC–PDA–ESI/MS/MS indicated that ellagi-
tannins were the most predominant components of all three methanol
fractions (Singab et al., 2011).
The secondary metabolites screening of E. camaldulensis leaf extracts
from Nigeria confirmed presence of tannin, saponins, and cardiac gly-
cosides (Ayepola and Adeniyi, 2008). Analyzed n-hexane, chloroform,
and methanol extracts of E. camaldulensis stem bark and leaf also grown
in Nigeria showed the presence of tannins and saponins in the stem bark
and in the leaf of E. camaldulensis with absence of alkaloids in all ex-
tracts (Adeniyi et al., 2009). Furthermore, the crude methanol leaf
extracts also from Nigeria contained in addition volatile oils and balsam
(gum) (Babayi et al., 2004). Similarly, the phytochemical screening of
ethanol, methanol, and petroleum ether leaf extracts from Nigeria
contained in moderate to high amount secondary metabolites: alka-
loids, saponins, tannins, flavonoids, steroid, carbohydrates, and cardiac
glycosides, and not anthraquinones (Chuku et al., 2016). Crude
V. Aleksic Sabo and P. Knezevic
methanol leaf extracts of E. camaldulensis from Iran had saponins, tan-
nins, volatile oils, and balsam (gum), while the components such as
anthraquinones, hydrolysable tannin, flavonoid, alkaloid, and glyco-
sides were not detected (Jouki and Khazaei, 2010); crude methanolic
leaves extract of E. camaldulensis from India contained anthraquinones,
flavonoids, saponins, and terpenoids, while alkaloids, cardiac glyco-
sides, and tannins were not detected (Singh and Thakur, 2016). Phy-
tochemical screening of the crude stem barks methanol extract of E.
camaldulensis from Bangladesh indicated presence of saponins, flavo-
noids, tannins, and also volatile oils, while anthraquinones, hydro-
lysable tannins, alkaloids, and glycosides were not present (Islam et al.,
2014). The polyphenolic composition (flavonoids and phenolic acids
and aldehydes) also was studied in the soluble fractions of the metha-
nolic extracts of Eucalyptus camaldulensis originating from two Spain
provinces, Huelva and Pontevedra: gallic, protocatechuic, vanillic and
ellagic acids, and protocatechic aldehyde were identified, along with
eriodictyol, quercetin, naringenin, vanillin, naringin, quercitrin, lu-
teolin, and kaempferol (Cadahia et al., 1997). Eucalyptus camaldulensis
extracts are generally rich in tannins which vary qualitatively and
quantitatively according to the origin of the samples, and consequently
protoanthocyanidin levels were influenced by the geographical origin
(Cadahia et al., 1997).
3. Antimicrobial effect of Eucalyptus camaldulensis extracts and
essential oils
Antimicrobial activity of E. camaldulensis EO and extracts are well
documented against many microorganisms listed in the Table 4. For
easier comparison, all data commented here for EOs were re-calculated
from microliter per milliliter or microgram/milligram per milliliter to
percentage (v/v or w/v), using the equitations presented in Fig. 2. We
considered here only minimal inhibitory and minimal bactericidal
concentrations, while results obtained using disc or agar diffusion
methods were not discussed. However, MIC/MBC results vary between
several micrograms to several milligrams. Such high variation does not
seem as real, even taking into account variation in oil composition, and
are rather a consequence of erroneous equalization of one microliter
and one microgram, or typographical errors. For instance, there are
some nonsense MICs, such as 2000 μL/mL, that is practically impossible
to obtain (Salem et al., 2015). In some manuscripts even a species was
not precisely indicated (Harkenthal et al., 1999; Karpanen et al., 2008;
Warnke et al., 2009; Tadtong et al., 2016) and these results were not
considered in the present review.
3.1. Antibacterial effect
Eucalyptus camaldulensis plant extracts and EOs were tested against
wide range of bacteria. The most frequently included Gram positive
bacterium in screening is S. aureus. Minimal inhibitory concentrations
in most studies were in range 0.07–0.5% indicating moderately high
activity against this bacterium. Beside S. aureus, antibacterial effect was
confirmed against B. subtilis (0.17–0.34%), M. luteus (0.2–0.4%), and S.
pyogenes (0.4–1.1%) (Rasooli et al., 2009; Akin et al., 2010; Knezevic
et al., 2016; Khubeiz et al., 2016; Reda et al., 2017; Ostad Asiaei et al.,
2018). Activity of EOs was examined aginst L. monocytogenes in only
one study and MIC was not obtained with the highest used concentra-
tion of 1.0%, and the same was reported for Enterococcus durans (Akin
et al., 2010).
Activity against Gram negative bacteria has been documented in a
greater extent and MICs for the most frequently used model organism E.
coli was in range 0.15–3.2%. Sensitivity of other enterobacteria is si-
milar, with MICs in range from 0.05 to 0.32% for K. pneumoniae
(Khubeiz et al., 2016; Ostad Asiaei et al., 2018), 0.16–0.32% for Sal-
monella enterica subsp. enterica serovar Typhimurium (Khubeiz et al.,
2016); 0.35–0.4% for serovars Typhi, Paratyphi, and 0.6% for S. En-
teritis (Ostad Asiaei et al., 2018). Other enterobacteria are similarily
419
Industrial Crops & Products 132 (2019) 413–429
sensitive: Shigella soneii was inhibited with 0.3% of EO (Ostad Asiaei
et al., 2018), while Proteus vilgaris was sensitive in a broad range from
0.25% (Ostad Asiaei et al., 2018) to more than 1.28% (Khubeiz et al.,
2016). The published data for P. aeruginosa are in the same range as for
P. vulgaris, which is not surprising, as both species are generally highly
resistant to antimicrobial agents. On the contrary, A. baumannii which
is also known to be multi-drug resistant, showed sensitivity to EO in a
range 0.05–0.1% (Knezevic et al., 2016). The lowest MIC among Gram
negative bacteria was recorded for a gastrointestinal pathogen V.
parachaemoliticus (0.01%) (Khubeiz et al., 2016).
Although the Gram positive bacteria are consider more sensitive to
EOs in comparison to Gram negative, it cannot be applied for essential
oils of E. camaldulensis, since the most sensitive bacteria are Gram ne-
gative – A. baumannii and V. parahaemolyticus. The minimal bactericidal
concentrations for most bacteria are equal to, or rarely up to 4 times
greater than MICs. This low range in MIC/MBC < 4 indicates that E.
camaldulensis EOs act as bactericidal agents (Pankey and Sabath, 2004).
Antibacterial effects of E. camaldulensis extracts have been examined
in a wider extent than essential oils. Inhibitory concentrations varied
depending on extraction method, plant properties, and model organism,
being in broad range from 0.08 μg/mL to 200 mg/mL. Crude aqueous
leaf extracts show lower activity against various bacteria (range
25–50 mg/mL) (Abubakar, 2010), while bark aqueous extracts were
more effective, with MIC from 0.1 mg/mL for Propionobacterium acnes
to 4.0 mg/mL for P. aeruginosa (Mabona et al., 2013). The antimicrobial
activity of methanol, ethanol or petroleum leaf extracts of E. camaldu-
lensis showed significant variation; for instance, MICs against B. subtilis
varied from 0.04 up to 200 mg/mL or against S. aureus 1.25–25 mg/mL
(Chuku et al., 2016; Ayepola and Adeniyi, 2008). For most examined
Gram negative bacteria MICs were in range 10–200 mg/mL, while P.
aeruginosa was even more susceptible (MICs 10–100 mg/mL). Similar or
better antibacterial effect showed acetone leaf extract, being active
against examined bacteria in range 15–50 mg/mL. The highest activity
was obtained with dichloromethen extracts, with MICs for Staphylo-
coccus spp. in range 0.25–1.0 mg/mL, and even lower for B. subtilis, P.
acnes and B. agri (MICs 0.10–0.79 mg/mL). Interestingly, generally
highly resistant M. tuberculosis was sensitive to methanol, n-hexane or
chloroform extract with MICs in range 0.004–0.064 mg/mL, while M.
bovis was slightly less sensitive with MICs 0.01–0.05 mg/mL (Lawal
et al., 2012; Gemechu et al., 2013).
The progresses made on the investigation of essential oils mode of
action, especially against bacterial cell targets, gave new perspectives in
this combat. The knowledge about the essential oils and their target(s)
on bacterial cell is crucial to understand which parts of bacterial cell are
affected. The essential oils antibacterial action is linked to oil hydro-
phobicity that increases cell permeability and consequent leakage of
cell constituents (Dorman and Deans, 2000; Lambert et al., 2001;
Helander et al., 1998; Turgis et al., 2009; Ultee et al., 2002; Faleiro,
2011). It is important to perceive that a disturbed cell structures may
affect stability of other cellular structures in a cascade type of action
(Carson et al., 2002). Essential oils have several target sites on bacterial
cells; however it seems that all are directly or indirectly connected to
the primary effect of essential oils on the bacterial envelopes. First of
all, EOs may cause cell wall and membrane disturbance (Lambert et al.,
2001; Oussalah et al., 2006), which further can lead to the significant
loss of intracellular ATP (Oussalah et al., 2006; Turgis et al., 2009),
induction the synthesis of heat shock proteins (Burt et al., 2007), pH
disturbance (Turgis et al., 2009; Oussalah et al., 2006), and in-
tracytoplasmic changes (e.g. coagulation, periplasmic space enlarge-
ment) (Becerril et al., 2007). Although the number of studies dealing
with the EO mechanisms of action is increasing, there are still many
questions to be answered before the precise mechanism is revealed.
This is at the same time one of the main limitations for EOs and extracts
wide usage as antimicrobials. Thus, the so far knowledge must be fur-
ther improved enabling the combat bacterial pathogens and its re-
sistance.
V. Aleksic Sabo and P. Knezevic Industrial Crops & Products 132 (2019) 413–429

Table 4
Antimicrobial activity of E. camaldulensis essential oils and extracts.
E. camaldulensis Microorganism Activitya Recalculated (%) References

MIC MBC MIC MBC

Essential oil Leaves essential oil (%, v/v) S. aureus 0.5 Akin et al. (2010)
from Northern Cyprus L. monocytogenes, E. durans, >1
Salmonella Typhi, E. coli, B.
subtilis, P. aeruginosa
Leaves essential oil (μg/mL) S. aureus, E. coli 8 0.0008 Lima et al. (2013)
from Iran
Leaves essential oil (μg/mL) S. aureus 3.9 3.9-7.8 0.00039 0.00039– Panahi et al.
from Iran 0.00078 (2011)
Leaves essential oil (μL/mL) Trichophyton sp. 2.5–5.0 0.27–0.53 Nasir et al. (2015)
from Ethiopia Microsporum, Candida 5.0 0.53
Rhodotorula
A. niger 2.5 0.27
E. coli, Shigella sp, Bacillus sp, 5.0 0.53
streptococci
Essential oil (μg/mL) from P. aeruginosa ATCC 27853 64 128 0.0064 0.0128 Owlia et al.
Iran (2009)
Leaves essential oil (%) from S.aureus, A. baumannii 0.1 0.2 Ostad Asiaei et al.
Iran E.coli 0.15 0.25 (2018)
P. vulgaris 0.25 0.35
S. sonnei 0.30 0.45
P. aeruginosa 0.2 0.40
K. pneumonia 0.05 0.15
S. eneterica serovar. Typhi 0.4 0.6
S. eneterica serovar. Paratyphi 0.35 0.45
S. eneterica serovar. Enteritidis, 0.6 0.8
Infantis
-1
Leaves essential oil (mg mL ) Streptococcus mutans, S. 1 2 0.1 0.2 Rasooli et al.
from Iran pyogenes (2009)
Leaves essential oil (mg mL-1) T. cucumeris, F. oxysporum, C. 5 0.5 Siramon et al.
from Thailand globosum (2013)
A. niger, C. cladosporioides, F. 10 1
palustris, P. citrinum, T.
versicolor
R. oryzae >10 >1
-1
Leaves essential oil (μl mL ) Reference Acinetobacter 0.5–1 0.7–4 0.05–0.1 0.07–0. 4 Knezevic et al.
from Montenegro baumannii and MDR clinical (2016)
strains
S. aureus ATCC 25923, E. coli 1 1–2 0. 1 0.1–0.2
ATCC 25922
Leaves essential oil (μl/mL) Fusarium sp. 7–8 8–10 0.7–0.85 0.85–1 Gakuubi et al.
from Kenya (2017)
Leaves essential oil (μl/mL) F. graminearum 2.5 0.27 Mehani et al.
from Algeria F. sporotrichioide 1.25 0.13 (2014)
Leaves essential oil (μg mL-1) S. aureus 1000 0.1 Chaves et al.
from Brazil E. coli >1000 >0.1 (2018)
Leaves essential oil from % of reduction El-Baz et al.
Egypt Rotavirus Wa strain 50 (2015)
Coxsackievirus B4 53.3
Herpes virus type 1 90
Adenovirus type 7 0
EOs in vegetable oil (mg/mL) Trypanosoma brucei brucei 100 10 Habila et al.
and Trypanosoma evansi (2010)
Leaves essential oil (mg/mL) S. aureus 0.2 0.8 0.02 0.8 Khubeiz et al.
from Syria B. subtilis 1.6–3.2 1.6–3.2 0.16–0.32 0.16–0.32 (2016)
M. luteus 0.2–0.4 0.4–1.6 0.02–0.04 0.04–0.16
S. pyogenes 0.4–0.8 0.8–1.6 0.04–0.08 0.08–0.16
K. pneumonia 1.6–3.2 3.2 0.16–0.32 0.32
V. parahaemolyticus 0.1 0.2 0.01 0.02
S. Typhimurium 1.6–3.2 3.2 0.16–0.32 0.32
E. coli 0.4–3.2 0.8–3.2 0.04–0.32 0.08–0.32
P. vulgaris, Ps. aeruginosa >12.8 >12.8 >1.28 >1.28
Leaves essential oil from Iran GI Katooli et al.
P. ultimum, R. solani, B. 100% (2011)
sorokiniana, C. gloeosporioides
P. digitatum, A. flavus 0%

Plant extract Leaves acetone extract (mg/l) MDR Staphylococcus aureus 20–50 0.002–0.005 Ibrahim et al.
from Nigeria (2014)
Leaves methanol extract MDR S. aureus 0.78 0.078 Reda et al. (2017)
(mg/mL) from Egypt MDR P. aeruginosa 3.12 0.312
Bark butanol extract (μg mL- Pectobacterium cartovorum 16 0.0016 EL-Hefny et al.
1
) from Egypt Ralstonia solanacearum 125 0.0125 (2017)
Agrobacterium tumefaciens 250 0.025
Dickeya spp. 500 0.05
(continued on next page)
420
V. Aleksic Sabo and P. Knezevic Industrial Crops & Products 132 (2019) 413–429

Table 4 (continued)

E. camaldulensis Microorganism Activitya Recalculated (%) References

MIC MBC MIC MBC

Leaves methanol extracts S. aureus, B. subtilis 5 0.5 Potdara et al.

(mg/mL) from India P. aeruginosa, S. eneterica 10 1.0 (2015)
serovar. Paratyphi P. vulgaris, 25 2.5
K.
pneumonia,

S. eneterica 50 5.0
serovar.
Typhi

E. coli 75 7.5
Leaves crude methanolic Dickeya solani, B. cereus 0.08 0.16 0.000008 0.000016 Elansary et al.
extracts (μg mL-1) from S. aureus 0.22 0.40 0.000022 0.00004 (2017)
Egypt
Leaves methanol, ethanol, P. aeruginosa 100 200 10 20 Chuku et al.
and petroleum ether extracts B. subtilis 50–200 200–400 5–20 20-40 (2016)
(mg mL-1) from Nigeria E. coli 50–200 100–200 5–20 10–20
Penicillium expansum 50–100 50–400 5–10 5–40
C. albicans 50–200 50–200 5–20 5–20
Leaves S. aureus, methicillin-resistant 0.063 0.0063 Takahashi et al.
methanol–dichloromethane S. aureus (2004)
extracts (mg mL-1) from B. cereus, E. faecalis, 0.125 0.0125
Japan Propionibacterium acnes,
Trichophyton mentagrophytes
E. coli, Pseudomonas putida, >0.250 >0.0250
Alicyclobacillus acidoterrestris
Leaves crude methanol B. subtilis, C. albicans, S. aureus 200 0.02 Babayi et al.
extracts (μg mL-1) from (ATCC103207), S. aureus (2004)
Nigeria (clinical)
E. coli (ATCC 10418), P. >200 >0.02
aeruginosa (ATCC 27853), S.
Typhi
Leaves methanol extract (mg Klebsiella spp, Salmonella Typhi, 10 1.0 Ayepola and
mL-1) from Nigeria P. aeruginosa Adeniyi (2008)
Leaves dichloromethane S. aureus 1.25 0.125
fraction (mg mL-1) from Yersinia enterocolitica 0.157 0.0157
Nigeria B. subtilis 0.04 0.004
Klebsiella spp, Salmonella Typhi, 10 1.0
P. aeruginosa
S. aureus, Yersinia enterocolitica 0.625 0.0625
B. subtilis 0.79 0.079
Leaves crude n-hexane Helicobacter pylori ATCC43504 <25 <0.0025 Adeniyi et al.
extracts; crude chloroform and ATCC47619 (2009)
leaves and steam extracts (μg
mL-1) from Nigeria
Steam crude n-hexane <12.5 <0.00125
extracts (μg mL-1) from
Nigeria
Leaves and steam bark Mycobacterium tuberculosis 4–52.2 0.0004–0.0064 Lawal et al.
hexane, chloroform, H37Rv (2012)
methanol extracts (μg/mL)
from Nigeria
Leaves methanol extracts M. tuberculosis 6.25–50 0.000625–0.005 Gemechu et al.
80% (μg/mL) from Ethiopia M. bovis 12.5–50 0.00125–0.005 (2013)
Leaves crude water extracts E. coli, Salmonella Typhi, S. 50 5.0 Abubakar (2010)
(mg/mL) from Nigeria aureus
Leaves crude ethanol extracts Proteus mirabilis, Klebsiella 25 2.5
(mg/mL) from Nigeria pneumoniae
Leaves crude acetone extracts E. coli, 50 5.0
(mg/mL) from Nigeria Salmonella Typhi, S. aureus, 25 2.5
Klebsiella pneumoniae
Proteus mirabilis 15 1.5
E. coli, Klebsiella pneumoniae 25 2.5
Salmonella Typhi, S. aureus, 15 1.5
Proteus mirabilis
Leaves essential oil (mg/mL) S. aureus 0.7 0.07 Reda et al. (2017)
from Egipt P. aeruginosa 3.12 0.312
Leaves DMSO extract (μg/ Newcastle Disease Virus (NDV) 50-500 0.005-0.05 Al-Hadid (2016)
mL) from Jordan
Leaves methanol extract (μg/ IC50 IC50 Abu-Jafar and
mL) from Israel Herpes simplex virus -1 0.1±0.08 0.3±0.02 0.00001±0.000008 Huleihel (2017)
Herpes simplex virus -2 1±0.03 0.00003±0.000002
Varicella-Zoster Virus 0.0001±0.000003
(continued on next page)

421
V. Aleksic Sabo and P. Knezevic Industrial Crops & Products 132 (2019) 413–429

Table 4 (continued)

E. camaldulensis Microorganism Activitya Recalculated (%) References

MIC MBC MIC MBC

Leaves methanol extracts Poliovirus type I neutralization index of Adeniyi et al.

from Nigeria one log and above (2015)
Coxsackievirus B
Echovirus
6

Bark n-butanol extract (μg P. carotovorum 16 0.0016 El-Hefny et al.

/mL) from Egypt R. solanacearum 125 0.0125 (2017)
A. tumefaciens 250 0.025
Dickeya spp. 500 0.05
Leaves methanol extracts Microsporum canis 0.8 6.4 0.08 0.64 Falahati et al.
(mg/mL) from Iran M. gypseum 1.6 3.2 0.16 0.32 (2005)
Tricophyton rubrum 1.6 1.6 0.16 0.16
T. schoenleinii 0.4 0.8 0.04 0.08
T. mentagrophytes 0.2 0.8 0.02 0.08
Epedermophyton floccosum 0.2 0.8 0.02 0.08
Bark dichloromethane: MICD:M MICAQ MICD:M MICAQ Mabona et al.
methanol extract; aqueous S. aureus (3 strains, including 0.25–1.0 0.5–0.63 0.025–0.1 0.05–0.0- (2013)
extract (mg/mL) from South MRSA) 63
Africa S. epidermidis 0.5 2.0 0.05 0.2
P. aeruginosa 2.0 4.0 0.2 0.4
C. albicans 0.5 2.0 0.05 0.2
Brevibacillus agri 0.25 0.20 0.025 0.02
Propionobacterium acnes 0.10 2.0 0.01 0.2
Trichophyton mentagrophytes 1.0 1.0 0.1 0.1
M. canis 4.0 2.0 0.4 0.2
Leaves methanol and Leishmania major IC50 IC50 Nosratabadi et al.
aqueous extracts (μg/mL) 586.2-1108.6 0.05862–0.11086 (2015)
from Iran
Leaves crude, Trichomonas vaginalis 100% growth inhibition with 12.5–50; 100% growth Hassani et
diethyl ether, 24–72 h inhibition with al. (2013)
ethyl acetate, 1.25–5%; 24–72 h
aqueous (mg/
mL) from Iran
Leaves wather Trichomonas vaginalis Dead for 72 h with 60-90 Dead for 72 h with Youse et
and ethanolic 0.006–0.009% al. (2012)
extracts (μg/
mL) from Iran
Leaves aqueous Trichomonas vaginalis Dead for 24 h with 500 Dead for 24 h with 50% Mahdi et
extract (mg/ al. (2006)
mL) from Iraq

a
MIC – minimal inhibitory concentration, MBC – minimal bactericidal concentration, GI – growth inhibition, IC50 - 50% inhibitory concentration.

Contemporary research of EOs and extracts are also focused on
mechanism that allows bacteria to regulate some physiological activ-
ities, such as virulence, competition amongst populations, motility,
sporulation, conjugation, antibiotic production, and biofilm formation
(Rodriguez-Garcia et al., 2014). This system of intercellular commu-
nication, quorum sensing (QS), is based on production of the signal
molecules, called autoinducers (Abraham et al., 2011). This commu-
nication system is relative to cell density and some compounds interfere
with the QS communication system and attenuating the bacterial pa-
thogenicity, in the phenomenon known as anti-QS compounds
(Abraham et al., 2011). There are reports of anti-QS activity of various
plants species EOs from genus Eucalyptus, such as Eucalyptus globulus L.
(Luís et al., 2016; Cervantes-Ceballos et al., 2015), Eucalyptus radiata D.
(Luís et al., 2016), Eucalyptus citriodora Hook., Eucalyptus smithii R. T.
Baker and Eucalyptus staigeriana F. Muell. ex Bailey (Luís et al., 2016).
Unfortunately, there is still no data regarding E. camaldulensis anti-QS
activity.
In one study, antibacterial and in vivo biofilm preventive efficacies
of E. camaldulensis oil were significantly higher than that of M. spicata
oil and chlorhexidine, suggesting that E. camaldulensis EO is capable of

Fig. 2. Schematic diagram for re-calculation from microliter or milligram per milliliter to percentage (v/v or w/v).

422
V. Aleksic Sabo and P. Knezevic
affecting biofilm formation (Rasooli et al., 2009). Considering the anti-
QS activity of above mentioned Eucalyptus species and also detected
antimicrobial and anti-biofilm effect of E. camaldulensis it can be as-
sumed that it possess similar or even higher anti-QS activity. However,
future detailed studies of anti-QS and anti-biofilm E. camaldulensis ac-
tivity are needed in order to confirm this assumption.
3.2. Antifungal effect
The E. camaldulensis leaves extracts and EOs have a potential as
antifungal agents. They are able to act as a moderate antifungal agent
against household molds, wood rot fungi (Siramon et al., 2013), and
phytopathogenic fungi (Gakuubi et al., 2017; Mehani et al., 2014).
Eucalyptus camaldulensis EO has been studied for antifungal activity
and was active in concentration 0.125–1.0% against most model or-
ganisms. The most sensitive seems to be F. sporotrichioides with MIC
0.125% (Mehani et al., 2014), while R. oryzae is the most resistant, as
1% of EO was ineffective against this fungus (Siramon et al., 2013). The
best known human and animal pathogenic yeast Candida sp. showed
considerable sensitivity to E. camaldulensis EO, with MIC approx. 0.5%
(Siramon et al., 2013; Nasir et al., 2015). Most EOs from Sardinia in-
hibited growth of A. niger and B. cinerea in concentration of 20 μL/plate,
while lower concentrations of the oils, such as 5 μL/plate were in-
effective (Barra et al., 2010).
It is interesting to notice that liposomes containing E. camaldulensis
EO were prepared (Moghimipour et al., 2012) for antifungal oil ac-
tivity. The particle size varied from 40.5 to 298 nm for the different
formulations, with approx. 95% of the essential oil entrapped. Inhibi-
tion of Microsporum canis, M. gypseum, Trichophyton rubrum, and T.
verrucosum was achieved with 125 μL, and the liposomal gel formula-
tion of the EO was proposed to improve antifungal activity.
Aqueous and organic extracts of E. camaldulensis have been reported
to have antifungal activity (Table 4). Methanol leaves extracts showed
inconsistent activity against C. albicans: in one study it was in range
50–200 mg/mL (Chuku et al., 2016), while in another it was 0.2 mg/mL
(Babayi et al., 2004), indicating difference in active concentration of
one thousand times. The bark methanole extract was active in con-
centration 0.5 mg/mL. Methanol leaf and bark extracts showed con-
siderable activity against dermatophytes: 0.8–1.6 mg/mL against Mi-
crosporum spp, 0.125–1.6 mg/mL against Trichophyton spp., and
0.2 mg/mL against Epidemophyton flocossum (Takahashi et al., 2004;
Falahati et al., 2005; Mabona et al., 2013).
Beside the antifungal activity of EOs and extracts of E. camaldulensis,
it is worth noticing that this species, along with E. blakelyi M., E.
gomphocephala A. DC, E. rudis Endl., and E. tereticornis Sm., is a reservoir
of an emerging pathogenic fungus – Cryptococcus gattii. This fungus is
usually related to tropic and subtropic regions, affecting the respiratory
and nervous systems of the immunocompetent humans and domestic
animals (Sorrell et al., 1996; Chakrabarti et al., 1997; Bielska and May,
2016; Roe et al., 2018). Similarily, C. neoformans var. grubii can be
isolated from flowers and bark of E. camaldulensis (Gugnani et al.,
2005). According to the literature, E. camaldulensis is natural reservoir
of Cryptococcus and is considered responsible for occurring crypto-
coccosis worldwide. In this context, it is interesting to observe that C.
neoformans is moderately sensitive to E. citriodora Hook EOs, with
MIC90 0.5% (wt/v) (Pattnaik et al., 1996; Luqman et al., 2008) or E.
globulus Labill. with MIC 0.13% (wt/v) (Suliman et al., 2010). Un-
fortunately, data on Cryptococcus sp. sensitivity to EOs and plant ex-
tracts of reservoir species of Eucalyptus, including E. camaldulensis, re-
mains unknown.
Although there are no reports regarding E. camaldulensis mode of
antifungal action, according to previous studies with other essential oils
and fungi, the plasma membrane and the mitochondria are the probable
antifungal targets of EOs. According to recent studies, the antifungal
activity of EOs results from its ability to disrupt the permeability barrier
of the plasma membrane and from the mitochondrial dysfunction-
423
Industrial Crops & Products 132 (2019) 413–429
induced ROS accumulation in A. flavus and C. albicans (Tian et al.,
2012; Chen et al., 2013). The exact mechanism of E. camaldulensis an-
tifungal activity is unknown and should be elucidated in the future.
3.3. Antiviral effect
Infections caused by viruses are very common and sometimes life
threatening, especially in immunocompromised patients and neonates
(Snoeck, 2000; Khan et al., 2005). Despite the recent significant pro-
gress in antiviral drug development, viral infections are considered as
one of the major causes of death worldwide (Müller et al., 2007; Meyers
et al., 1982). Thus, novel natural antiviral agents need to be found
urgently. Many natural products possess antiviral activity and some of
them are already in use for treatment of human viral infections with
both RNA and DNA viruses (e.g. myricetin against coronavirus, linalool,
urosolic acid, and apigenin against coxsackievirus, quercetin and nar-
asin against denge virus, curcumin against hepatitis B and C virus) (Lin
et al., 2014; Kitazato et al., 2007). Numerous secondary plant meta-
bolites such as essential oils, flavonoids, saponins, tannins, alkaloids,
lignans, terpenes, and phenolic acids express significant antiviral ac-
tivity against different viruses (Jassim and Naji, 2003; Chiang et al.,
2003; Sanchez Palomino et al., 2002). Recently few studies confirmed
the E. camaldulensis EOs and plant extracts antiviral activity (Table 4).
Eucalyptus camaldulensis EOs reduce coxackie B4 and rotavirus Wa
multiplication for 50%, herpes simplex virus 1 for 90%, but have no
effect on adenovirus 7 multiplication (El-Baz et al., 2015). Similarly,
methanolic extracts showed 50% inhibition of HSV 1 and 2 in con-
centration 0.1–0.3 μg/mL, and against Varicella zoster virus at con-
centration 1.0 μL/ml (Abu-Jafar and Huleihel, 2017). Dimethyl sulf-
oxide (DMSO) extracts inhbit multiplication of animal New Castle virus
in concentration range 50–500 μg/mL (Al-Hadid, 2016). Antiviral ac-
tivity has been observed for E. camaldulensis methanolic extracts against
polio virus, coxsackie B, and echovirus 6 (Adeniyi et al., 2015). The
data on antiviral activity of E. camaldulensis EO and extracts, although
scarce, indicate their great potential, and necessity for further studies in
this context.
Despite the fact that many plant extracts and essential oils were
previously reported for their antiviral activities, the mechanism of ac-
tion still remains poorly understood. There are many factors that in-
fluencing the EOs mode of action, which should be taken in con-
sideration when antiviral activity of plant antimicrobials is examined.
One of such factors is the difference between enveloped and non-en-
veloped viruses, because the observed antiviral effect has usually been
greater for enveloped viruses (Yamada et al., 2009; Siddiqui et al.,
1996). In majority of the studies dealing with antiviral mode of action,
the focus has been on either the inhibition of viral adsorption to host
cells or examination of the plant antimicrobials effectiveness against
intracellular virus multiplication (Gilling et al., 2014). So, most com-
monly described modes of antiviral actions are virus inactivation and
the impaired virus adsorption to host cells, which is often difficult to
distinguish. Like in other antimicrobial modes of action, antiviral me-
chanism is also dependent on EOs or extracts compounds activity. This
is one more factor that should be considered, because it affects the final
antiviral mechanism of action. For some compounds the potential me-
chanism is reported, i.e. carvacrol acts directly upon the virus capsid
and subsequently the nucleic acid (Gilling et al., 2014). When EOs
antiviral mechanism of action is considered, EOs may cause the loss of
the viral capsid integrity, ultimately leading to exposure of the viral
genome. In addition, some EOs subsequently act directly upon the viral
nucleic acid (DNA or RNA). According to one study, E. camaldulensis
EOs may be promising antiviral agent against RNA viruses with no ef-
fect against DNA virus (El-Baz et al., 2015). Also, with shorter periods
of exposure to the antimicrobial, the virus is able to adsorb specifically
to host cells; however, it may or may not be able to cause successful
infection depending upon the integrity of the viral genome. On the
other hand, after exposure to some EOs virus capsid and genome
V. Aleksic Sabo and P. Knezevic
remains intact. These antimicrobials appear to exert their antiviral ef-
fect by coating the capsid and thereby preventing specific adsorption of
the virus to host cells (Gilling et al., 2014). Although, there are still no
reports regarding E. camaldulensis EOs antiviral mode of action, there
are some promising results about its antiviral effect (Table 4). These
promising results represent foundation for further research and poten-
tial application of E. camalulensis EOs as potent antiviral agent.
3.4. Antiprotzoal effect
Among all deverse benefitial activities, E. camaldulensis expresess
also an antiprotozoal effect as well. The reports regarding this effect are
listed in Table 4. Eucalyptus camaldulensis methanolic and aqueous ex-
tracts were active against Leishmania major with IC50 values (50% in-
hibitory concentration) 586.2 ± 47.6 and 1108.6 ± 51.9 μg/mL, re-
spectively (Nosratabadi et al., 2015). This was characterized as
moderate leishmanicidal activity, but considering fact that present
therapy consist antimony compounds which are expensive, toxic, and
drug resistance is prevalent, E. camaldulensis plant extract derivatives
represent safe, inexpensive, and promising alterantive solution.
There are also several reports regarding antiprotozoal activity
against Trichomonas vaginalis, a causative agent of trichomoniasis which
is the most prevalent nonviral sexual infection. The first report on E.
camaldulensis aqueous extract anti-trichomonas activity showed that
extract was active at concentration 500 mg/mL after 24 h (Mahdi et al.,
2006). However, recent studies reported better effect of the E. cama-
ladulensis extracts (Hassani et al., 2013; Youse et al., 2012). Five dif-
ferent E. camaldulensis leaves extracts including total extract, diethyl
ether, chloroform, ethyl acetate, and water fractions were active in
concentration range 12.5–50 mg/mL, with growth inhibiton achived
after 24–72 h (Hassani et al., 2013). Ethyl acetate fraction showed the
highest percentage of growth inhibition with the lowest concentration
(12.5 mg/mL) after 24 and 48 h (Hassani et al., 2013). Even better anti-
trichomonas activity was detedted for wather and ethanolic extracts,
where concentration 60–90 μg/mL killed Trichomonas vaginalis for 72 h
(Youse et al., 2012). The mainstay medication for trichomoniasis is
metronidazole; however some resistant strains to this treatment have
been detected making these results of E. camaldulensis anti-trichomonas
activity very promising as antiprotozoal agent.
Except extracts, significant antiprotozoal activity was reported for E.
camaldulensis EOs. The essential oils were found to possess anti-
trypanosomal activity in vitro in a dose-dependent manner in a short
time. The decrease of Trypanosoma evansi number over time was
achieved in doses of 400 mg/mL for 3 min, 200 mg/mL for 4 min, and
100 mg/mL for 15 min. Against Trypanosoma brucei brucei EOs was
more potent in the concentration of 400 mg/mL, decreasing the number
of parasite for 3 min, 200 mg/mL for 4 min, and 100 mg/mL for 11 min
(Habila et al., 2010). Such prompt decrease in parasite number in the in
vitro tests for both Trypanosoma brucei brucei and T. evansisuggests that
the EOs kill the parasites efficiently, but by an unknown mechanism.
There are suggestions of some potential mechanisms of antiprotozoal
EOs action. The activity of EOs could be due to the hydrophobic nature
of the cyclic hydrocarbons, which allow EOs to interact with the pro-
tozoans causing conformational changes in the parasite membrane
structure, resulting in the loss of membrane stability (Calsamiglia et al.,
2007). The essential oils also can act by inhibiting some key enzymes in
the parasite glycolytic pathway (Smith-Palmer et al., 2004). Further-
more, some EOs components inhibit acetylcholinesterase activity and
act on other vulnerable sites, such as cytochrome p450 (Maciel et al.,
2010). This multicomponent nature of plant EOs is an advantage for
several target sites on protozoans, which is of great importance.
3.5. Antimicrobial activity of E. camaldulensis Dehn. vs. other species of
genus Eucalyptus
It was shown that EOs of E. camaldulensis are more potent against B.
424
Industrial Crops & Products 132 (2019) 413–429
subtilis, S. aureus, E. coli, P. aeruginosa, S. lutea, and P. carotovorum in
comparison to EOs from E. gomphocephala DC., but not against A. tu-
mefaciens. Furthermore, the E. camaldulensis var. obtuse showed even
better effect than E. camaldulensis (Salem et al., 2015). Among seven
examine essential oils of various Eucalyptus species, EO of E. camaldu-
lensis was among the best against B. subtilis, A. niger, and R. solani, but
not against E. coli and S. aureus (Ghaffar et al., 2015). Similar activity of
E. camaldulensis and E. torelliana F. Muell. was recorded for extracts
against six strains of H. pylori (Adeniyi et al., 2009).
It is worth to notice that among 132 extracts from 42 plants growing
in southern Africa, E. camaldulensis bark extract showed considerable
activity against bacteria and fungi, with an exception against P. aeru-
ginosa and M. canis (Mabona et al., 2013). Finaly, in many studies re-
garding antimicrobial activity of various Eucalyptus species, E. ca-
maldulensis was not always included (Ashour, 2008; Safaei-Ghomi and
Ahd, 2010; Mulyaningsih et al., 2010; Elaissi et al., 2012; Sebei et al.,
2015).
4. Interaction of Eucalyptus camaldulensis Dehn. EOs/extracts and
other antimicrobial agents
Due to rapid emerging of microbial resistance to conventional
drugs, the necessity for efficient solution(s) is rising. The main strategy
represents finding new antimicrobial agents; however another strategy
goes in the direction of reducing degree of bacterial resistance and/or
bacterial re-sensitization to conventional antibiotics. This can be
achieved using combined therapies. The most of the tested combina-
tions are dual combinations, but the combinations of three, four or
more agents also can be efficient (Lesjak et al., 2016). This is in line
with antimicrobial efficiency and potential of EOs and plant extracts
which are complex mixtures of numerous compounds. Combination
strategy could be very promising regarding the diversity of agents that
could be tested: (1) conventional non-antimicrobial agent (e.g. anti-in-
flammatory or anti-psychotic drug) + conventional antimicrobial agent
(antibiotic); (2) conventional antimicrobial agent (antibiotic) + natural
antimicrobial agent (essential oil, plant extract, bacteriophage, anti-
microbial peptide ect.); (3) conventional antimicrobial agent (anti-
biotic) + single compound isolated from natural antimicrobial agent
(with previously confirmed antimicrobial activity); (4) combination of
two or more single compounds isolated from natural antimicrobial
agents (with previously confirmed antimicrobial activity).
Being a plant with already detected and evaluated antimicrobial
activity, it can be assumed that E. camaldulensis also have a potential in
combined therapy. This assumption is confirmed in some studies in vitro
and in vivo (Table 5). Eucalyptus camaldulensis EOs and extracts in vitro
reduced resistance of MDR A. baumannii in combination with conven-
tional antibiotics: β-lactams, ciprofloxacin, gentamicin, polymyxin B
(Knezevic et al., 2016). Similarily, the increase of β-lactamase produing
MRSA and E. coli sensitivity to cephalexin, cefuroxime, amoxicillin, and
ampicillin has been obtained through combination with E. camaldulensis
EOs (Chaves et al., 2018). Synergistic activity has been recorded be-
tween E. camaldulensis plant extracts and gentamicin or ceftriaxone
against MDR S. aureus and P. aeruginosa (Reda et al., 2017; Ibrahim
et al., 2014), as well as in combination with ampicillin against S. aureus
(Ibrahim et al., 2014).
Furthermore, the combination of E. camaldulensis extract with an-
other plant extract Psidium guajava L. was also efficient against MDR
bacteria (Bala et al., 2014). Except antibacterial activity, other activ-
ities of E. camaldulensis extracts in combination were detected and
characterized as efficient. Antiviral activity was confirmed for the
combination of E. camaldulensis 80% methanol leaves extract and acy-
clovir against herpes simplex virus -1 and -2 and varicella-zoster virus
(Abu-Jafar and Huleihel, 2017). Similaily, the combination of Annona
senegalensis L. leaf methanol extract and E. camaldulensis extract effi-
ciently cured in vivo albino mice infection with parasite Trypanosoma
brucei brucei (Lafia strain) (Kabiru et al., 2012). All these data are very
V. Aleksic Sabo and P. Knezevic Industrial Crops & Products 132 (2019) 413–429

promising, but the methods used in different studies and results inter-

Reda et al. (2017)

Bala et al. (2014)

pretation vary (Table 5). As a consequence, the data should be taken

Huleihel (2017)
Knezevic et al.

Abu-Jafar and

Ibrahim et al.
with precautions, as can not be compared and properly discussed. To

Chaves et al.

Kabiru et al.
Reference

avoid this problem, the testing combinations of different agents should

(2016)

(2018)

(2012)

(2014)
be conducted using standardized methods, such as time-kill method
(CLSI, 1999; Verma, 2007), checkerboard method (Verma, 2007;
EUCAST, 2000), Chou-Talalay method (Chou, 2010) or Boik method

Synergism (only the combination 1:1 resulted

in the complete clearance of parasites from
Combining the essential oil with β-lactams

(Boik, 2010). All these methods possess some shortfalls, such as time-

infection, comparing to single agent (10-

Significant inhibition (˜75%) of the viral
reduced the resistance of tested strains

consuming, labor-intensive, limitations regarding the number of the

agents in combination, etc. Unfortunately, there is no one gold standard
for synergy testing and prior the further application of different phy-
tochemicals, this issue should be overcome.
the circulation of animal)
Synergism (FICI ≤ 0.5)
Effect of combination

5. Conclusion

Summarizing the available data on antimicrobial properties of

Synergism

Synergism

Synergism
Eucalyptus camaldulensis essential oil and extracts, it is obvious that this
plant is a valuable source of phytotherapeutics. The essential oil, as well
20%)

as leaf and bark extracts are particularly valuable as antibacterial, an-

tiviral, and antifungal agents, and their antiprotozoal activity should
The MIC of the antibiotics were determined in the presence

200 mg/kg bodyweight/day (for 21 days) of crude methanol

Treating the cells with different combinations of the extract

extracts of A. senegalensis and E. camaldulensis combinations

and absence of sub-inhibitory concentrations (125 μg mL−1)

and acyclovir at the time and post-infection with the virus

Agar disc diffusion method was employed as described by

Kirby and Bauer (1966), adopted by Yushau et al. (2009)

not be neglected taking into account current therapy cost, toxicity, and
Two-dimensional checkerboard method (Verma, 2007)

protozoal growing resistance. Some E. camaldulensis plant character-

istics such as easy cultivation, wide distribution by plantation, and
rapid growth additionally support further examination of antimicrobial
activity, in order to enhance the commercial production of E. ca-
of the essential oil (Coutinho et al., 2010)

in ratios 1:1, 1:2, and 2:1, respectively.

maldulensis based pharmaceuticals. The future studies should be fo-

cused on determination of the mechanisms of antimicrobial activity,
paticularly potential anti-biofilm and anti-QS effects, as well as the
activity enhancement in combination with other available agents.
and Bashir et al. (2011)
Lajubutu et al. (1995)

Acknowledgments
Collins et al. (1995)

This study was supported by the Ministry of Education, Science and

Technological Development of the Republic of Serbia, grant OI 172058.
Method

The authors acknowledge Prof. Stephen D. Hopper (School of

Agriculture and Environment, The University of Western Australia) for
Eucalyptus camaldulensis image and Prof. Ljiljana Knezevic (Faculty of
Reference Acinetobacter baumannii and MDR

In vitro infection of Vero cells with Herpes

Sciences, University of Novi Sad) for the language revision.

simplex virus -1 Herpes simplex virus -2

Trypanosoma brucei brucei (Lafia strain)

S. aureus 55 (MRSA) E. coli (all strains

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